Digging deep into “dirty” drugs – modulation of the methylation machinery

“Histone octamers consist of eight proteins, consisting of two copies of each of the core histones H2A, H2B, H3 and H4 (Luger et al., 1997).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“DNMT3A and 3B exhibit predominant denovo methyltransferase activity, initiating DNA methylation marks on unmethylated DNA (Okano et al., 1999) (Figure 2A and C), whereas DNMT1 is exclusively involved in the The green field demarks methylation, whereas the red field demarks occurring demethylation.”

“TET proteins can also drive the 5-mC oxidation process further to produce 5formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) as reaction products (Pfeifer et al., 2014; Tahiliani et al., 2009) (Figures 1A-V, 1A-VI, 2B and D).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“EZH2 is only active when it is associated with the other PRC2 core components: embryonic ectoderm development (EED), suppressor of zeste 12 homolog (SUZ12) and retinoblastoma binding protein 4 (RBBP4); (Kogure et al., 2013; Margueron & Reinberg, 2011) (Figure 2C).”

“These epigenetic changes persist through cell divisions for the duration of the cell’s life, and may be inherited over multiple generations (Bird, 2007).”

“…in primary AML patient samples and murine models of acute leukemia, partially through the down-regulation of the critical oncogenes, MYC and Bcl2 (Chen et al., 2014; Dawson et al., 2014; Delmore et al., 2011; Fiskus et al., 2014b; Mertz et al., 2011; Valent & Zuber, 2014; Zuber et al., 2011).”

“Almost 35 years later, both drugs have shown significant clinical benefits with high overall response rates and improved overall survival in patients with myeloid malignancies (Fenaux et al., 2009, 2010; Kantarjian et al., 2012; Pleyer et al., 2013, 2014a, b).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“…of crosstalk between DNA methylation and histone post-translational modifications, whereby histone modifications are thought to provide labile transcriptional repression, and DNA methylation results in more permanent gene silencing (Cedar & Bergman, 2009; Ikegami et al., 2009; Raynal et al., 2012).”

“(Marcucci et al., 2010; Mardis et al., 2009).”

“HMTs methylate arginine and lysine residues of histones, and are involved in the regulation of a wide range of processes including gene activity, chromatin structure and epigenetic memory (Martin & Zhang, 2005).”

“IDH gain-of-function mutations and TET2 loss-of-function mutations (which are mutually exclusive in AML) affect the same pathway, and ultimately induce the same global promoter hypermethylation phenotype (Dang et al., 2009; Madzo et al., 2013; Ward et al., 2010).”

“…in primary AML patient samples and murine models of acute leukemia, partially through the down-regulation of the critical oncogenes, MYC and Bcl2 (Chen et al., 2014; Dawson et al., 2014; Delmore et al., 2011; Fiskus et al., 2014b; Mertz et al., 2011; Valent & Zuber, 2014; Zuber et al., 2011).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“Several small molecule inhibitors of EZH2 have been developed, and shown to induce tumor regression in hematologic tumor models (Knutson et al., 2012; McCabe et al., 2012; Qi et al., 2012).”

“Dysfunction of BET-proteins has been linked to cancer (Filippakopoulos et al., 2012), and members of the BET family have recently been identified as targets for modulation of chromatin dynamics.”

“1, have been associated with impaired survival in MDS (Bejar et al., 2011) and CMML (Grossmann et al., 2011).”

“The HDAC-inhibitors vorinostat and romidepsin have received FDA approval for cutaneous T-cell lymphoma, and the large number of clinical trials with these substances has been summarized by others (Copeland et al., 2009; Falkenberg & Johnstone, 2014; Johnstone, 2002).”

“DNMT2 methylates tRNAASP and DNMT3L is catalytically inactive, but formation of a DNMT3L–DNMT3A-complex results in enhancement and recruitment of DNMT3A activity to histone H3 tails, when H3K4 is not methylated (Ooi et al., 2007) (Figure 2A).”

“Thus, aberrant DNA methylation patterns are no longer reproduced in the daughter cells, leading to DNA demethylation and subsequent gene (re-)activation (Christman, 2002; Stresemann & Lyko, 2008).”

“The HDAC-inhibitors vorinostat and romidepsin have received FDA approval for cutaneous T-cell lymphoma, and the large number of clinical trials with these substances has been summarized by others (Copeland et al., 2009; Falkenberg & Johnstone, 2014; Johnstone, 2002).”

“UHRF1 has an affinity for hemimethylated CpG DNA and recruits DNMT1 to ensure that the sequence becomes completely methylated following replication in order to maintain the epigenetic inheritance of DNA methylation (Bostick et al., 2007; Sharif et al., 2007) (Figure 3A).”

“Mammalian cells contain five DNMT isoforms, but only DNMT1, 3A and 3B methylate DNA (Bourc’his et al., 2001; Goll et al., 2006).”

“After binding to methylated CpG, MeCP2 recruits and forms the MeCP2/Sin3A/HDAC1 co-repressor complex to silence transcription via histone deacetylation (Nan et al., 1997) (Figure 2A).”

“These regions are usually non-methylated in normal cells, whereas aberrant hypermethylation is found in approximately 1.5% of the CpG-islands (on average 600 of 45 000) in the genome of malignant cells, and may show nonrandom and tumor-type specific patterns (Costello et al., 2000).”

“Loss of TET2 leads to increased self-renewal capacity of hematopoietic stem cells, expansion of myeloid precursors and myeloid transformation in mice (Moran-Crusio et al., 2011).”

“UHRF1 has an affinity for hemimethylated CpG DNA and recruits DNMT1 to ensure that the sequence becomes completely methylated following replication in order to maintain the epigenetic inheritance of DNA methylation (Bostick et al., 2007; Sharif et al., 2007) (Figure 3A).”

“Measurement of 5-hmC levels in myeloid malignancies may thus prove a valuable diagnostic and prognostic tool to individually tailor therapies and assess therapeutic response (Ko et al., 2010).”

“demethylation reaction (Klose et al., 2006) (Figure 2D).”

“, 2010), and wild-type EZH2 is commonly overexpressed in a variety of cancers (Bracken et al., 2003), only one report of EZH2 overexpression in MDS exists (Xu et al.”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“Mutations in other enzymes involved in the citric acid cycle, such as fumarate hydratase (FH) and succinate dehydrogenase (SDH), have been observed in solid tumors, and result in accumulation of fumarate and succinate, both of which are competitive inhibitors of TET proteins (Xiao et al., 2012).”

“Almost 35 years later, both drugs have shown significant clinical benefits with high overall response rates and improved overall survival in patients with myeloid malignancies (Fenaux et al., 2009, 2010; Kantarjian et al., 2012; Pleyer et al., 2013, 2014a, b).”

“…in primary AML patient samples and murine models of acute leukemia, partially through the down-regulation of the critical oncogenes, MYC and Bcl2 (Chen et al., 2014; Dawson et al., 2014; Delmore et al., 2011; Fiskus et al., 2014b; Mertz et al., 2011; Valent & Zuber, 2014; Zuber et al., 2011).”

“As such, 5- hmC levels correlate positively with reduced methylation, reduced recruitment of repressive proteins, and elevated levels of transcription of 5-hmC-marked genes (Jin et al., 2011; Song et al., 2011).”

“EZH2 expression is essential for cancer survival (Varambally et al., 2008).”

“Altered 2-HG/a-KG levels observed in cells with mutant IDH1/2 additionally result in the inhibition of the jumonjidomain-containing (JMJC) family of HDMs, another group of a-KG-dependent enzymes, which demethylate H3K9 and H3K26 (Chowdhury et al., 2011) (Figures 1B-III and 2A).”

“As mentioned above for UHRF1, this is mediated by recruitment of HMT activity by MeCP2, and results in particular results in increased H3K9 methylation (Fuks et al., 2003).”

“Almost 35 years later, both drugs have shown significant clinical benefits with high overall response rates and improved overall survival in patients with myeloid malignancies (Fenaux et al., 2009, 2010; Kantarjian et al., 2012; Pleyer et al., 2013, 2014a, b).”

“This suggests an important role for non-renal clearance (Chabot et al., 1983; Stresemann & Lyko, 2008).”

“As such, DOT1L inhibitors have been developed and shown to selectively kill cells bearing the MLL gene translocation in vitro, and to prolong survival in mouse and rat MLL xenograft models of AML (Daigle et al., 2011, 2013).”

“Recently, a core set of (aberrantly) methylated genes predictive of survival and specific for distinct biological AML subtypes was identified (Figueroa et al., 2010).”

“The dysregulation of HOX genes has recently been implicated in leukemogenesis, where they have been shown to support the immortalization of leukemic cells, both as chimeric partners in fusion genes, and also when overexpressed in their wild-type form (Alharbi et al., 2013; Shah & Sukumar, 2010).”

“…occur in 8% of MDS patients and in 23% of newly diagnosed AML patients, and have been implicated in disease pathogenesis, and as prognostic parameters for worse overall survival (Hopfer et al., 2009; Ley et al., 2010; Ribeiro et al., 2012; Thol et al., 2011a; Walter et al., 2011; Yan et al., 2011).”

“…marks in succeeding DNA-replication cycles (Pastor et al., 2011; Valinluck et al., 2004); (iii) 5-hmC blocks the binding of MBPs, in particular MeCP2 or MBD3, which usually confer transcriptional silencing (Ko et al., 2010; Pastor et al., 2011; Valinluck et al., 2004; Yildirim et al., 2011).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“Several small molecule inhibitors of EZH2 have been developed, and shown to induce tumor regression in hematologic tumor models (Knutson et al., 2012; McCabe et al., 2012; Qi et al., 2012).”

“(Marcucci et al., 2010; Mardis et al., 2009).”

“…marks in succeeding DNA-replication cycles (Pastor et al., 2011; Valinluck et al., 2004); (iii) 5-hmC blocks the binding of MBPs, in particular MeCP2 or MBD3, which usually confer transcriptional silencing (Ko et al., 2010; Pastor et al., 2011; Valinluck et al., 2004; Yildirim et al., 2011).”

“We summarize current knowledge on the involvement of the epigenetic machinery in the development of MDS/AML and their potential modulation by azanucleosides (if known); we also discuss which of these molecules may be additional therapeutic targets in MDS/AML, thus representing potential novel combination partners for…”

“…delta; Tc1, Type 1 CD8+ T cells; Th1, Type 1 T helper cell; Th17, Type 17 T helper cell; Tregs, regulatory T cells; U, uridine; UHRF1, ubiquitin-like with PHD and ring finger domains 1. transcriptional activator essential for hematologic differentiation and myeloid development (Tenen et al., 1997).”

“…the prognostic relevance of these mutations in MDS, CMML and AML remains unclear, and may be disease-specific, as currently existing data are inconsistent (Abdel-Wahab et al., 2009; Kosmider et al., 2009a, b; Liu et al., 2013; Metzeler et al., 2011b; Pollyea et al., 2011; Wang et al., 2013a).”

“HDACs are implicated in cancer through their aberrant recruitment and silencing of tumor suppressor genes.”

“As such, DOT1L inhibitors have been developed and shown to selectively kill cells bearing the MLL gene translocation in vitro, and to prolong survival in mouse and rat MLL xenograft models of AML (Daigle et al., 2011, 2013).”

“TET2 knockout mice develop a disease with MDS-like features (Li et al., 2011).”

“TET2 mutations occur in 20–25% of MDS, 7–23% of AML and up to 53% of CMML patients (Kosmider et al., 2009b; Shih et al., 2012; Yamazaki et al., 2012).”

“Similar to the effect of aforementioned EZH2 mutations, ASXL1-loss results in compromised function of PCR2, with ensuing loss of the transcriptionally repressive H3K27 trimethylation, leading to up-regulation of HOXA genes (Abdel-Wahab et al., 2012).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“%) seems to be sufficient to inactivate more than 95% of DNMT1 Creusot et al. (1982) 4 The depletion of DNMT1 occurs even in the presence of a potent inhibitor of DNA-synthesis.”

“In addition, the current conception of the incompletely understood phenomenon of long range gene silencing is summarized.”

“Systemic formulations have not yet reached clinical trials (Mochly-Rosen et al., 2012).”

“Several small molecule inhibitors of EZH2 have been developed, and shown to induce tumor regression in hematologic tumor models (Knutson et al., 2012; McCabe et al., 2012; Qi et al., 2012).”

“…2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al., 2013; Bejar et al., 2011; Inoue et al., 2013; Itzykson et al., 2013; Metzeler et al., 2011a; Patnaik et al., 2013, 2014).”

“…occur in 8% of MDS patients and in 23% of newly diagnosed AML patients, and have been implicated in disease pathogenesis, and as prognostic parameters for worse overall survival (Hopfer et al., 2009; Ley et al., 2010; Ribeiro et al., 2012; Thol et al., 2011a; Walter et al., 2011; Yan et al., 2011).”

“The commonly accepted mechanism of action of HMAs is based on the initial report that incorporation of these structurally similar cytosine analogues into DNA inhibits the capacity of DNMTs to methylate DNA (Santi et al., 1984).”

“Global methylation does not seem to differ between responders and non-responders to AZA or DAC (Daskalakis et al., 2002; Fandy et al., 2009).”

“HATs confer the acetylation of lysine residues on the N-terminus of histones, which is generally associated with active gene transcription (Yang, 2004) (Figure 2B and D).”

“…the onset of myeloproliferative AML in mice in co-operation with the transcription factor HOXA9, which is involved in stem cell maintenance and hematopoietic differentiation, thus proving oncogenic potential (Argiropoulos & Humphries, 2007; Chaturvedi et al., 2013; Nakamura et al., 1996).”

“Mutant IDH1 greatly accelerated the onset of myeloproliferative AML in mice in co-operation with the transcription factor HOXA9, which is involved in stem cell maintenance and hematopoietic differentiation, thus proving oncogenic potential (Argiropoulos & Humphries, 2007; Chaturvedi et al., 2013; Nakamura et al., 1996…”

“However, the presence of mutated TET2 has been associated with response to AZA and DAC in MDS and CMML by several (Braun et al., 2011; Itzykson et al., 2011; Traina et al., 2014; Voso et al., 2011), but not all groups (Pollyea et al., 2011).”

“The HDAC-inhibitors vorinostat and romidepsin have received FDA approval for cutaneous T-cell lymphoma, and the large number of clinical trials with these substances has been summarized by others (Copeland et al., 2009; Falkenberg & Johnstone, 2014; Johnstone, 2002).”

“The most accepted model is that direct physical interaction between promoters and their distant distal enhancers (highly enriched for the permissive H3K4me and H3K27ac marks) is brought about by chromatin ‘‘hopping’’ (Phair et al., 2004) or ‘‘looping’’ (Doyle et al.”

“…marks in succeeding DNA-replication cycles (Pastor et al., 2011; Valinluck et al., 2004); (iii) 5-hmC blocks the binding of MBPs, in particular MeCP2 or MBD3, which usually confer transcriptional silencing (Ko et al., 2010; Pastor et al., 2011; Valinluck et al., 2004; Yildirim et al., 2011).”

“As such, 5- hmC levels correlate positively with reduced methylation, reduced recruitment of repressive proteins, and elevated levels of transcription of 5-hmC-marked genes (Jin et al., 2011; Song et al., 2011).”

“Mutations in TET1 and TET2 have been observed in MDS, CMML and AML (Dolnik et al., 2012; Lorsbach et al., 2003).”

“Altered micro-RNA expression is frequently observed (Jongen-Lavrencic et al., 2008; Vasilatou et al., 2013), and deregulation of certain microRNAs has been implicated in disease pathogenesis (Raynal et al.”

“CTCF binding sites can act as nucleosome repositioning anchors, and thus represent a powerful means to regulate gene transcription (Fu et al., 2008).”

“…of all analyzed genes) are frequently hypermethylated in MDS, and that hypermethylation across the genome correlates with poor prognosis and transformation to AML, as well as AML relapse (Figueroa et al., 2009; Jiang et al., 2009; Kroeger et al., 2008; Maegawa et al., 2014; Zhao et al., 2014).”

“Interestingly, both of these proteins seem to be relevantly involved in the pathogenesis of MDS and AML, and mutually exclusive mutations in components of the cohesion complex or its partner CTCF have been reported in 8% of MDS patients (Kon et al., 2013).”

“The cohesion-complex is involved in chromosome pairing, DNA repair, and re-establishment of transcriptional regulation after cell division via ‘‘bookmarking’’ of transcription factor binding sites (Yan et al., 2013).”

“PKCs are regarded as oncogenes and as participants in carcinogenesis and leukemogenesis (Koivunen et al., 2006; Steinberg, 2004).”

“…such as EZH2 and additional sex combs like transcription regulator 1 (ASXL1), have recently been detected in MDS and AML, and are described in more detail below in ‘‘HMTs in MDS/AML’’ (Boultwood et al., 2010; El-Sharkawi et al., 2014; Nikoloski et al., 2010; Thol et al., 2011b; Wang et al., 2013b).”

“The production of 5-hmC results in DNA demethylation and increased gene expression via three mechanisms: (i) active demethylation through the activation of AID (Figure 1A-III), which results in degradation of 5- hmC to 5-hm-uracil (5-hmU), which in turn activates the base excision repair (BER) pathway for further degr…”

“…depletion, DNA hypomethylation and DNA damage induction), distinctly different effects in their action on sensitivity/cell viability (AZA had a greater effect on reduction of cell viability), total protein synthesis (reduced only by AZA), and gene expression were reported (Hollenbach et al., 2010).”

“In vitro data suggest synergism between the EZH2 inhibitor 3-deazaneplanocin-A (DZNep) and DAC and/or HDAC inhibitors in targeting AML cells (Fiskus et al., 2009; Momparler et al., 2012, 2014; Zhou et al., 2011).”

“This simultaneous inactivation of several genes occurs via an as yet unknown mechanism has also been found in MDS, where the presence of CIMP was associated with rapid progression to AML and shorter survival (Shen et al., 2010).”

“The dysregulation of HOX genes has recently been implicated in leukemogenesis, where they have been shown to support the immortalization of leukemic cells, both as chimeric partners in fusion genes, and also when overexpressed in their wild-type form (Alharbi et al., 2013; Shah & Sukumar, 2010).”

“…of all analyzed genes) are frequently hypermethylated in MDS, and that hypermethylation across the genome correlates with poor prognosis and transformation to AML, as well as AML relapse (Figueroa et al., 2009; Jiang et al., 2009; Kroeger et al., 2008; Maegawa et al., 2014; Zhao et al., 2014).”

“In addition, the current conception of the incompletely understood phenomenon of long range gene silencing is summarized.”

“AZA significantly down-regulated genes involved in the cell cycle, cell division and mitosis, whereas DAC preferentially up-regulated genes involved in cell differentiation (Flotho et al., 2009; Hollenbach et al., 2010).”

“…2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al., 2013; Bejar et al., 2011; Inoue et al., 2013; Itzykson et al., 2013; Metzeler et al., 2011a; Patnaik et al., 2013, 2014).”

“BET inhibitors attenuate cell growth and survival in primary AML patient samples and murine models of acute leukemia, partially through the down-regulation of the critical oncogenes, MYC and Bcl2 (Chen et al., 2014; Dawson et al., 2014; Delmore et al., 2011; Fiskus et al., 2014b; Mertz et al., 2011; Valent & Zuber, 20…”

“However, the presence of mutated TET2 has been associated with response to AZA and DAC in MDS and CMML by several (Braun et al., 2011; Itzykson et al., 2011; Traina et al., 2014; Voso et al., 2011), but not all groups (Pollyea et al., 2011).”

“In 1964, the azanucleosides 5-azacitidine (AZA) and 20-deoxy-5-azacitidine (decitabine, DAC) were developed as classical cytostatic agents (Sorm et al., 1964).”

“Global methylation does not seem to differ between responders and non-responders to AZA or DAC (Daskalakis et al., 2002; Fandy et al., 2009).”

“…2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al., 2013; Bejar et al., 2011; Inoue et al., 2013; Itzykson et al., 2013; Metzeler et al., 2011a; Patnaik et al., 2013, 2014).”

“TET2 mutations occur in 20–25% of MDS, 7–23% of AML and up to 53% of CMML patients (Kosmider et al., 2009b; Shih et al., 2012; Yamazaki et al., 2012).”

“…such as EZH2 and additional sex combs like transcription regulator 1 (ASXL1), have recently been detected in MDS and AML, and are described in more detail below in ‘‘HMTs in MDS/AML’’ (Boultwood et al., 2010; El-Sharkawi et al., 2014; Nikoloski et al., 2010; Thol et al., 2011b; Wang et al., 2013b).”

“…2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al., 2013; Bejar et al., 2011; Inoue et al., 2013; Itzykson et al., 2013; Metzeler et al., 2011a; Patnaik et al., 2013, 2014).”

“Results on the prognostic effects of IDH mutations in these diseases are divergent (Green et al., 2010, 2011; Patnaik et al., 2012).”

“Global DNA hypomethylation, as well as localized hypermethylation of genes, particularly of those encoding tumor suppressor genes, is a hallmark of most types of cancer (Feinberg & Tycko, 2004; McCabe et al., 2009).”

“PRDM factors act as HMTs, and function in developmental contexts, in which they drive and maintain cell state transitions (Hohenauer & Moore, 2012), and have also been implicated in methylation of non-histone targets (Fog et al., 2012).”

“…such as EZH2 and additional sex combs like transcription regulator 1 (ASXL1), have recently been detected in MDS and AML, and are described in more detail below in ‘‘HMTs in MDS/AML’’ (Boultwood et al., 2010; El-Sharkawi et al., 2014; Nikoloski et al., 2010; Thol et al., 2011b; Wang et al., 2013b).”

“PKCs are regarded as oncogenes and as participants in carcinogenesis and leukemogenesis (Koivunen et al., 2006; Steinberg, 2004).”

“…occur in 8% of MDS patients and in 23% of newly diagnosed AML patients, and have been implicated in disease pathogenesis, and as prognostic parameters for worse overall survival (Hopfer et al., 2009; Ley et al., 2010; Ribeiro et al., 2012; Thol et al., 2011a; Walter et al., 2011; Yan et al., 2011).”

“This process is referred to as ‘‘histone coding’’ (de la Cruz et al., 2005).”

“Maximal effects were observed at day 15, but methylation levels returned to near-baseline level by the end of each cycle (day 28) (Garcia-Manero et al., 2011).”

“Overexpression, amplification and mutations in all three catalytically active human DNMT isoforms have been described in numerous cancers (Miremadi et al., 2007).”

“TET2 mutations occur in 20–25% of MDS, 7–23% of AML and up to 53% of CMML patients (Kosmider et al., 2009b; Shih et al., 2012; Yamazaki et al., 2012).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“…such as gemcitabine (Marce et al., 2006), fludarabine (Molina-Arcas et al., 2003) and cytarabine (AraC; Hubeek et al., 2005), as well as the DNMT inhibitors DAC and AZA, suggesting their potential role as predictive biomarkers for clinical response (Damaraju et al., 2012; Qin et al., 2009).”

“DNA methylation and histone modification are epigenetic mechanisms that result in altered gene expression and cellular phenotype.”

“However, serial in vitro testing with 24 different HDAC-inhibitors efficiently reactivated various genes silenced by DNA hypermethylation, without affecting the latter, but this effect was transient (2 weeks) (Raynal et al., 2012).”

“…2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al., 2013; Bejar et al., 2011; Inoue et al., 2013; Itzykson et al., 2013; Metzeler et al., 2011a; Patnaik et al., 2013, 2014).”

“Both drugs are rapidly cleared from systemic circulation: plasma half-life 51.5 to 1.8 h, as compared to a half-life of 7–21 h at 37 C in neutral aqueous solutions in vitro (Liu et al., 2006; Yoo et al., 2007; Zhao et al., 2004).”

“The powerful antioxidant resveratrol which is extracted from grape skins, and present in both whole grapes and red wine, has recently been discovered to reduce DNMT1 and/or DNMT3B levels in breast cancer cell lines (Mirza et al., 2013), and also in tumor tissue in murine models of breast cancer (Qin et al.”

“It must further be considered that global methylation levels in blood DNA may vary in relation with age, gender, alcohol drinken, white blood cell count, as well as with the method used for methylation detection (Maegawa et al., 2014; Zhu et al., 2012).”

“In vitro data suggest synergism between the EZH2 inhibitor 3-deazaneplanocin-A (DZNep) and DAC and/or HDAC inhibitors in targeting AML cells (Fiskus et al., 2009; Momparler et al., 2012, 2014; Zhou et al., 2011).”

“Thus, a rational to combine inhibitors of CDA with HMA exists (Karahoca & Momparler, 2013).”

“Although PKCd modulators have been assessed in vitro and in murine models as a targeted cancer therapy, clinical trials have thus far only been performed with topical formulations in actinic keratosis, for which approval was gained.”

“Mutant IDH1 greatly accelerated the onset of myeloproliferative AML in mice in co-operation with the transcription factor HOXA9, which is involved in stem cell maintenance and hematopoietic differentiation, thus proving oncogenic potential (Argiropoulos & Humphries, 2007; Chaturvedi et al., 2013; Nakamura et al., 1996…”

“cells, and converted AML into an MDS/myeloproliferative neoplasm-like disease (Tanaka et al., 2012).”

“PRDM factors act as HMTs, and function in developmental contexts, in which they drive and maintain cell state transitions (Hohenauer & Moore, 2012), and have also been implicated in methylation of non-histone targets (Fog et al., 2012).”

“However, the presence of mutated TET2 has been associated with response to AZA and DAC in MDS and CMML by several (Braun et al., 2011; Itzykson et al., 2011; Traina et al., 2014; Voso et al., 2011), but not all groups (Pollyea et al., 2011).”

“Micro-RNAs are recognized as one of the major pillars that regulate gene expression and have been associated with most cellular functions, as well as with normal and malignant hematopoiesis (Vasilatou et al., 2010).”

“Mutations within all individual protein components of the cohesin-complex have been described, are mutually exclusive, and occur in 4–21% of MDS and AML patients (Leeke et al., 2014; Malcovati et al., 2014; Thol et al., 2014).”

“lation patterns during DNA replication (Spada et al., 2007).”

“Thus, in B-cells, AID is required for the generation of immunoglobulin diversity after V(D)J recombination (Honjo et al., 2004), an effect which is desired.”

“LSD1 inhibition resulted in increased methylation of H3K4, resulting in gene activation (Fiskus et al., 2014c).”

“The mixed lineage leukemia (MLL) gene, located on chromosome 11q23, is a recurrent locus of chromosomal translocation involved in leukemogenesis (Marschalek, 2011).”

“…al., 1982; Ghoshal et al., 2002, 2005), and DNMT1 depletion occurs even in the absence of (i) DNA replication and cell division (Datta et al., 2012; Easwaran et al., 2004; Ghoshal et al., 2005; Gius et al., 2004; Reither et al., 2003), and/or (ii) incorporation of AZA into DNA (Aimiuwu et al.,…”

“In contrast, 80–90% of AZA is converted to ribonucleoside triphosphate by uridine–cytidine kinase (UCK), and is incorporated into RNA, leading to inhibition of protein synthesis (Li et al., 1970; Van Rompay et al., 2001) (Figure 4).”

“Gene promoter hypermethylation leads to recruitment of co-repressors, altered chromatin structure, and ultimately transcriptional gene silencing (Lopez-Serra & Esteller, 2008).”

“In addition, the current conception of the incompletely understood phenomenon of long range gene silencing is summarized.”

“…in gene expression and DNMT1 degradation can also occur independently of cell division and in the absence of DNA replication Ghoshal et al. (2005), Gius et al. (2004) 5 Although all three DNMTs have the capacity to bind HMA-incorporated DNA, only DNMT1 is rapidly, selectively and completely…”

“ASXL1 mutations result in severe myelodysplasia and MDS-like disease in mice (Inoue et al., 2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al.”

“Mutations in other PRC2 components are rare in myeloid malignancies, but have recently been reported for SUZ12 and EED in MDS (Puda et al., 2012; Score et al., 2012).”

“Keywords Acute myeloid leukemia, azacitidine, decitabine, DNA methylation, epigenetics, hypomethylating agents, myelodysplastic syndrome, transcriptional regulation History Received 12 October 2014 Revised 26 November 2014 Accepted 1 December 2014 Published online 8 January 2015”

“Of interest, nucleophosmin 1 (NPM1) has recently been shown to inhibit BRD4, and NPM1 mutations abrogate this function.”

“…2013; Wang et al., 2014), occur in 49% of CMML, 5% of MDS and 14% of AML patients, and have been associated with poor prognosis (AbdelWahab & Levine, 2013; Abdel-Wahab et al., 2013; Bejar et al., 2011; Inoue et al., 2013; Itzykson et al., 2013; Metzeler et al., 2011a; Patnaik et al., 2013, 2014).”

“In addition, the current conception of the incompletely understood phenomenon of long range gene silencing is summarized.”

“We summarize current knowledge on the involvement of the epigenetic machinery in the development of MDS/AML and their potential modulation by azanucleosides (if known); we also discuss which of these molecules may be additional therapeutic targets in MDS/AML, thus representing potential novel combination partners for…”

“Total genomic 5-mC levels decreased on average by 14% in AML patients treated with DAC (Klco et al., 2013; Yang et al., 2006), and global reductions in methylation were also observed in isolated AML blasts (Claus et al., 2013).”

“transformation to AML in murine models (Sashida et al., 2014), or resulted in induction of differentiation in leukemic”

“However, apart from tyrosine kinase inhibitors targeting the fusion protein Bcr-Abl, drugs with such a narrow target spectrum have resulted in limited clinical benefits.”

“In particular, Jak2 inhibitors are effective, even in patients not harboring these mutations (Cervantes, 2014; de Lavallade et al., 2013; Ferreira & Harrison, 2014; Rosenthal & Mesa, 2014).”

“…of all analyzed genes) are frequently hypermethylated in MDS, and that hypermethylation across the genome correlates with poor prognosis and transformation to AML, as well as AML relapse (Figueroa et al., 2009; Jiang et al., 2009; Kroeger et al., 2008; Maegawa et al., 2014; Zhao et al., 2014).”

“Even for drugs thought to selectively target a certain oncogenic protein, such as Bcr-Abl or janus kinase 2 (Jak2), many ‘‘off-target’’ molecules are being found, as scientists dig deeper into their mechanisms of action.”

“In general, it is thought that methylation is responsible for creating a long-lasting state of transcriptional repression, which is preceded by repressive histone modifications such as H3-acetylation, H3K27-trimethylation or demethylation of permissive H3K4me3 (Ng et al., 2007).”

“…KENbox, which is a signature sequence for proteasomal degradation, located near the N-terminus of DNMT1 (but not DNMT3A/B) Datta et al. (2012), Ghoshal et al. (2002, 2005) 6 Dose-dependent degradation of DNMT1 still occurs in cells expressing aberrant DNMT1 that has lost the ability to bind…”

“New methods, such as single-cell DNA-methylation analyses, may be more appropriate (Lorthongpanich et al., 2013).”

“Three classes of enzymes are known to antagonize histone methylation: (i) peptidylarginine deiminase, (ii) lysine specific demethylase 1 (LSD1), and the Jumonji C domain family of histone demethylases (JHDMs) (Anand & Marmorstein, 2007).”

“However, clinically relevant results are yet to be demonstrated (Garcia-Manero, 2012; Quintas-Cardama et al., 2011; Stintzing et al., 2011).”

“Selective small molecule JHDM inhibitors are currently being developed (Luo et al., 2011; Xu et al., 2013), but have not yet reached clinical trials.”

“Recently, a core set of (aberrantly) methylated genes predictive of survival and specific for distinct biological AML subtypes was identified (Figueroa et al., 2010).”

“Several single locus studies have demonstrated promoter hypermethylation and silencing of isolated genes in MDS and AML, often in association with progressive disease, with e.g. p15(INK4B) being a prominent example (Christiansen et al., 2003; Tien et al., 2001).”

“, 2013), and deregulation of certain microRNAs has been implicated in disease pathogenesis (Raynal et al., 2012; Zardo et al., 2012).”

“Results on the prognostic effects of IDH mutations in these diseases are divergent (Green et al., 2010, 2011; Patnaik et al., 2012).”

“In AML (and more rarely in MDS and CMML), several HATs (EP300, CREBBP, MYST3 and MYST4) are commonly involved in chromosomal translocations, which are involved in leukemogenesis through aberrant acetylation of all core histones, as well as on various nonhistone proteins (Camos et al., 2006; Murati et al., 2004; Pattab…”

“LSD1 inhibition resulted in increased methylation of H3K4, resulting in gene activation (Fiskus et al., 2014c).”

“DCK deficiency has been reported as a major mechanism of resistance to the nucleoside analogues DAC, Ara-C and gemcitabine (Galmarini et al., 2003; Qin et al., 2011; Saiki et al., 2012; Veuger et al., 2003).”

“Results on the prognostic effects of IDH mutations in these diseases are divergent (Green et al., 2010, 2011; Patnaik et al., 2012).”

“AZA has recently been shown to be a potent and specific inhibitor of RRM2 in a murine AML model, AML cell lines, as well as in primary bone marrow cells from AML patients (Aimiuwu et al., 2012).”

“Retroviral overexpression of CDA caused significant resistance to DAC in vitro (Eliopoulos et al., 1998), and increased CDA expression/activity in human males in vivo has been linked with a reduced half-life of AZA and DAC, and possibly worse outcomes in MDS patients (Mahfouz et al., 2013).”

“UHRF1 also interacts with HDAC1 and recruits it to methlyated tumor suppressor genes (Jeanblanc et al., 2005) (Figure 3A).”

“This has been further underlined by intriguing observations that HDAC inhibitors can reactivate silenced genes without affecting their hypermethylated promoter state (Raynal et al., 2012).”

“The latter methylates CgP-islands in promoter regions with PU.1 binding sites (e.g. of p16INK4A (Kihara-Negishi et al., 2001; Suzuki et al., 2006) (Figure 2A)).”

“…direct T-cell stimulatory effects, upregulation of T-cell co-stimulatory molecules CD80 and CD86 on dendritic cells, and induction of anticancer humoral immune responses with enhanced antibody dependent cellular cytotoxicity (Ersvaer et al., 2007, 2010; Hampson et al., 2005; Olsnes et al., 2009).”

“In this regard, a very recent genome-wide methylation study demonstrated that differential methylation in AML was most pronounced in genomic areas far from CpG-islands, so-called ‘‘open sea regions’’ (Qu et al., 2014).”

“…on the involvement of the epigenetic machinery in the development of MDS/AML and their potential modulation by azanucleosides (if known); we also discuss which of these molecules may be additional therapeutic targets in MDS/AML, thus representing potential novel combination partners for HMAs.”

“…CREBBP, MYST3 and MYST4) are commonly involved in chromosomal translocations, which are involved in leukemogenesis through aberrant acetylation of all core histones, as well as on various nonhistone proteins (Camos et al., 2006; Murati et al., 2004; Pattabiraman et al., 2014; Shigeno et al., 2004).”

“TET2 mutations occur in 20–25% of MDS, 7–23% of AML and up to 53% of CMML patients (Kosmider et al., 2009b; Shih et al., 2012; Yamazaki et al., 2012).”

“…of crosstalk between DNA methylation and histone post-translational modifications, whereby histone modifications are thought to provide labile transcriptional repression, and DNA methylation results in more permanent gene silencing (Cedar & Bergman, 2009; Ikegami et al., 2009; Raynal et al., 2012).”

“…resveratrol which is extracted from grape skins, and present in both whole grapes and red wine, has recently been discovered to reduce DNMT1 and/or DNMT3B levels in breast cancer cell lines (Mirza et al., 2013), and also in tumor tissue in murine models of breast cancer (Qin et al., 2014).”

“MBPs bind to methylated CgP-islands and mediate transcriptional repression of affected genes (Parry & Clarke, 2011).”

“TET proteins can also drive the 5-mC oxidation process further to produce 5- formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) as reaction products (Pfeifer et al., 2014; Tahiliani et al., 2009) (Figures 1A-V, 1A-VI, 2B and D).”

“DCK deficiency has been reported as a major mechanism of resistance to the nucleoside analogues DAC, Ara-C and gemcitabine (Galmarini et al., 2003; Qin et al., 2011; Saiki et al., 2012; Veuger et al., 2003).”

“(DAC/5-AZA) inhibit DNA methyltransferases (mainly DMNT1 and DMNT3) resulting in the re-expression of tumor-suppressor genes [419, 420].”

“In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), for instance, promoters of tumor suppressor genes are frequently found to be hypermethylated (Pleyer and Greil 2015).”

“Other replication-independent mechanisms have been proposed as well and are reviewed elsewhere [14].”

“Eventually, this leads to global DNA hypomethylation, which enables the re-expression of genes, among others tumor suppressor genes, causing an overall anti-neoplastic effect [28].”

“Decitabine is initially phosphorylated by deoxycytidine kinase rather than uridine-cytidine kinase and, unlike azacitidine (incorporated into both RNA and DNA), is only incorporated into DNA [9, 10, 15, 16].”

“Despite significant research efforts, the mechanism of action of these drugs is not completely understood [343], and re-expression of Wnt may be one of the many reasons why they are clinically effective.”

“in patients with myeloid malignancies (Pleyer and Greil, 2015).”

“In our study, treatment with AZA failed to induce SVCT expression, probably based on their different mode of action [38].”

“Mechanistically, the anti-neoplastic effect of HMAs is thought to mainly result from a global DNA demethylation allowing for re-expression of silenced genes, including tumor suppressor genes and testis cancer antigens (Steele et al., 2009; Pleyer & Greil, 2015).”

“Associations with pretreatment expression of multiple GMDs are in agreement with the multifaceted actions of epigenetic agents which affect multiple molecular components [1, 100, 103].”

“DNMTis have a wide range of cellular effects, through phosphorylation and DNA binding, inducing DNA hypomethylation, apoptosis, or activation of specific genes, such as tumor suppressor genes, to exert anti-tumor effects (143).”

“Given the lack of a curative option for most patients and similar survival, the toxicity profile of HMA makes this option more attractive [57].”

“This mechanism of action makes DNMT2 susceptible to the action of DNA methyltransferase inhibitors ribo- (5-azacytidine or 5-AZAC) or deoxyribo- (deoxy5-azacytidine or DAC5) while ensuring that the function of other RNA methyltransferases in the cell remain unperturbed [25,26].”

“5-azacytidine is mainly incorporated into RNA, affecting RNA synthesis and stability, and thereby also protein synthesis, and part of 5-azacytidine is reduced to 5AzadC which forms DNA adducts, causing mutations, double strand breaks and apoptosis, as reviewed [8].”

“apoptotic pathway were in agreement with earlier reports [1, 32, 53].”

“5-aza-dC is now utilized as epigenetic medicine to recover the expression of tumor suppressor genes by reducing DNA methylation [41].”

“Recently, a growing number of studies on decitabine and azacitidine suggest separate mechanisms of action of these drugs [19].”

“DAC is currently used as a clinical treatment for myelodysplastic syndrome and acute myeloid leukemia [32].”

“Azacitidine (5′-azacytidine) is a clinically approved hypomethylating agent that inhibits DNA methylation of tumor suppressor genes, enhancing their transcription, and has shown significant clinical benefits in patients with myeloid malignancies [35, 51, 52].”

“In addition, low dose HMAs also trigger type 1 interferon production and RNase L–mediated cell death by reactivating endogenous retroviruses [89–93].”

“…(1) Catalytic inhibition of DNMTs induces genome-wide hypomethylation, leading to chromatin structural remodeling and increased genomic instability; (2) During DNA replication, DNMTi incorporates into nascent strands as abnormal deoxycytidine analogs, generating single-strand DNA breaks [59].”

“Novel therapies aimed to treat cancer by using histone methylation drugs show positive results (Harb-de la Rosa et al., 2015; Pleyer and Greil, 2015; Castilho et al., 2017; Choi et al., 2017; Curry et al., 2018).”

“Although AZA and DAC have been clinically used for more than half a century, and despite decades of research to define their mechanisms, little is known about their precise mechanisms of action [32].”

“The non-selective HDAC inhibitor vorinostat and the DNMT1 inhibitor azacytidine induced concerted transcriptional effects in multiple HDAC and SIRT genes, consistent with broad epigenetic effects of both agents [25,36,74–77].”

“The nucleoside analogs 5-azacytidine (ZCyt, Azacytidine), 5-aza-2 ′ - deoxycytidines (ZdCyd), and 5-fluoro-2 ′ -deoxycytidine (FdC, Decitabine) can be incorporated into RNA and DNA, thereby interfering with DNA, RNA, and protein biosynthesis [148].”

“In academic research a preferred term is off-target drugs, nevertheless a definition proposed by Pleyer et al [32] for dirty drugs summarizes the concept as a drug that targets either “several structurally related proteins, a cellular complex composed of several proteins and/or molecules that govern several downstream…”

“When DAC is incorporated into DNA, it prevents DNA from becoming methylated and inhibits DNA methyltransferases [4].”

“Nevertheless, further analyses using ChIP-seq data, promoter motif enrichment, or histone modification signatures are needed to validate the significance of HDAC4 in melanoma prognosis.”

“DAC is known to be passively incorporated into the DNA and to irreversibly bind DNMT1, thereby blocking maintenance methylation in proliferating cells, but also known to be incorporated into the DNA by active mechanisms [72].”

“Targeting these upstream pathways can reduce DNMT1 abundance or activity, thereby promoting DNA demethylation.”

“Predictors of positive responses to DMTIs is include (1) doubling of PLT count, (2) mutated TET2, (3) mutated EZH2, (4) Phosphoinositide-phospholipase C beta hypomethylation, and (5) low serum level of micro-RNA-21 [96, 104, 105].”